Reversible two-step unfolding of heme-human serum albumin: a (1)H-NMR relaxometric and circular dichroism study.
Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme-HSA has been investigated by (1)H-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate (thus fully saturating all available fatty acid binding sites in serum heme-albumin), 1.0 M guanidinium chloride induces some unfolding of heme-HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K (d) = (1.2 +/- 0.1) x 10(-6) M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K (d) = (3.4 +/- 0.1) x 10(-5) M]. Further addition of guanidinium chloride (up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme-HSA in the presence of myristate.
Fanali
Gabriella
G
Dipartimento di Biologia Strutturale e Funzionale, and Centro di Neuroscienze, Università dell'Insubria, Via Alberto da Giussano 12, 21052, Busto Arsizio (VA), Italy.
De Sanctis
Giampiero
G
Gioia
Magda
M
Coletta
Massimo
M
Ascenzi
Paolo
P
Fasano
Mauro
M
D001665:Binding Sites; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D006418:Heme / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D019814:Myristic Acid / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D011522:Protons; D012709:Serum Albumin / Q000737:chemistry*
D011522:Protons; D012709:Serum Albumin; D019814:Myristic Acid; D006418:Heme; D019791:Guanidine